1. Field of the Invention
A method and kit to monitor and for diagnosis of cellular immune deficiencies.
2. Description of Related Art
A variety of clinical tests have been used to evaluate cellular immune responses in diseases involving congenital or acquired cellular immune deficiency (see in general Stites et al. (eds), Basic and Clinical Immunology (8th Edition), Appleton and Lange, Norwalk, Conn. (1994)). Primary cellular immune deficiency such as DiGeorge Syndrome is associated with the lack of a thymus and thus T-lymphocytes in blood and tissues. Secondary cellular immune deficiencies occur in a variety of conditions (see Table I), perhaps most notably in cancer and human immunodeficiency virus (HIV) infections. In these diseases, impairment of T-lymphocyte functions (proliferation, cytokine production, cytotoxicity, help and suppression) are often manifest early and followed by a loss of T-lymphocytes in blood (lymphocytopenia) and lymphoid tissues. Often, monocyte/macrophage defects occur which may contribute to impairment of cellular immunity (CMI) also referred to as delayed-type hypersensitivity (DTH).
DTH is frequently tested clinically with the use of skin tests to recall antigens such as purified protein derivative (PPD), tetanus toxoid, candida and other antigens. These tests are not generally standardized. One test device, the Merieux Multitest device, has been employed; however, it is expensive ( greater than $90/test) and insensitive and prone to false negative reaction. Perhaps the most useful test historically, particularly in cancer, has been primary sensitization with a new antigen like dinitrochlorobenzene (DNCB), followed by a challenge. The DNCB skin test has been useful to predict survival in patients with human cancer i.e. patients with a good response to DNCB will live longer and have better survival rates with conventional therapy then those who have a poor response. It is, however, difficult to perform and there are concerns regarding possible toxicity, and cross-sensitizing (Stites, et al, ibid at page 196) and is thus seldom used.
T-lymphocyte (CD3, CD11) and subsets (CD4, CD8) counts using cytofluorometry (FACS) are now performed frequently on patients with HIV infection and are considered to be an important prognostic predictor of the outcome of this disease. FACS analyses of T-lymphocyte and subsets are expensive tests and generally only available in the industrialized countries.
Historically, physicians have performed routinely a complete blood count (CBC) and differential. With these data the white blood cell count (WBC) and the percent of lymphocytes are determined; however, in general, the WBC and percent lymphocyte are not multiplied to yield a lymphocyte count (normally around 2000 mm3). Further, approximately 20% of circulating lymphocytes are B-lymphocytes and these values have been determined to vary little throughout life and during various disease. Thus, physicians are generally unaware that lymphocyte counts of  less than 1500 mm3 reflect mainly T-lymphocytopenia approaching clinical significance.
Where analyzed, T-lymphocytopenia as occurs in cancer, cancer therapy, irradiation, and HIV infection generally reflects greater early losses of CD4 than CD8 cells. Thus, one can determine a lymphocyte count and infer with a great degree of accuracy that if it is significantly low (i.e.  less than 1500 mm3) then T-lymphocytopenia is present and cellular immunity may be impaired.
A variety of in vitro tests of T-lymphocyte function have been developed including lymphoproliferative responses to mitogens like phytohemagglutinin (PHA) or Concanavalin A (Con A) , interleukin (IL) production like IL-2 and gamma interferon (IFN-xcex3), cytotoxicity for target cells, etc. These tests are expensive, cumbersome, and performed only at academic centers where appropriate research laboratories are present.
Clinical medicine needs not only new approaches to the diagnosis of cellular immune deficiency but also to monitor the effect of immunotherapy. Historically, natural killer (NK) cells have been employed to monitor the effect of recombinant interferon alpha (rIFN-xcex1) therapy, yet in fact clinical responses have not correlated with NK cell number or activity. T-lymphocyte counts have responded to intravenous therapy with high dose rIL-2; however the response is little or not at all with subcutaneous therapy with low doses of rIL-2. Thus, new approaches are needed for the diagnosis and monitoring of CMI in human patients with cancer and other diseases in which cellular immune deficiencies are common.
The use of a natural cytokine mixture (NCM) has proven effective in the treatment of cellular immune deficiency relating to age and stress (U.S. Pat. No. 5,632,983).
The clinical application of this NCM in the immunotherapy of four patients with squamous cell head and neck cancer (HandN SCC) induced significant immune regressions in three of four patients (Hadden et al. xe2x80x9cInterleukins and Contrasuppression Induce Immune Regression of Head and Neck Cancerxe2x80x9d Int. Arch. Otolaryngol., 120:395-403, 1994). In this study, a DTH intracutaneous skin test was performed. Three patients who were lymphocytopenic responded to NCM therapy with increased T-lymphocyte counts, including the skin test negative patient; thus, the skin test did not appear to predict the immunorestorative response to NCM.
It would be useful to effectively analyze DTH in the patient with cellular immune deficiency and to monitor the effect of immunotherapy designed to correct this deficiency.
According to the present invention, a method and kit for determining candidates for immunotherapy, for monitoring the effect of immunotherapy and analysis of cell mediated immunity functionality in a patient is provided. The method includes performing two intracutaneous skin tests and reading the skin test after twenty-four hours. One skin test is the administration of a mitogen such as phytohemagglutinin (PHA), concanavalin A (ConA), pokeweed antigen (PWA) and other mitogens as known in the art. The PHA skin test responses reflects the ability of the T-lymphocytes which are present to react to PHA and to release cytokines like IL-2 and induce a monocyte/macrophage infiltration leading to the DTH dermal reaction which is observed in the skin test characteristic of the afferent limb response of the immune system. The NCM (mitogen- stimulated natural cytokine mixture) skin test reflects the ability of preformed T-cell cytokines to induce the monocyte/macrophage accumulation characteristic of the efferent limb response.
The kit contains the appropriate mitogen and NCM to be used in the skin tests.
In other words, the present invention provides a method to monitor patients with cellular immune deficiency by the steps of determining the result of intracutaneous skin tests with a mitogen such as PHA and with natural cytokine mixture (NCM) and the result of blood lymphocyte counts (with or without T-lymphocyte and subset enumeration) to yield a composite xe2x80x9cthree-dimensional viewxe2x80x9d of CMI including T-lymphocyte number and function (afferent limb) and cytokine production and action on monocytes and macrophages (efferent limb)